TIMP2 Mediates Endoplasmic Reticulum Stress Contributing To Sepsis-associated Acute Kidney Injury

Objective Tissue inhibitor of metalloproteinase 2(TIMP2)is a biomarker for early identification of sepsis-associated acute kidney injury(AKI),but its biological function in septic AKI is unclear.Endoplasmic reticulum stress,a pathological state indicating the imbalance of endoplasmic reticulum protein homeostasis in cells,has been demonstrated to be involved in the development of multiple renal diseases,while its role in sepsis-associated AKI has not been fully elucidated.In this study,we intended to clarify the expressions of TIMP2 and endoplasmic reticulum stress-related proteins in septic AKI,and to demonstrate the biological roles they play in the progression of sepsis-associated AKI.In addition,we aimed to verify whether there is a relationship between TIMP2 and endoplasmic reticulum stress,and to investigate the molecular mechanisms of underlying the regulation of TIMP2 and endoplasmic reticulum stress proteins.Methods The cecal ligation and puncture(CLP)model of sepsis was performed on mice.We employed fluorescence quantitative polymerase chain reaction(q-PCR),western blotting and immunostaining assays to explore the expressions of TIMP2 and molecules in ER stress.Besides,conditional knockout(CKO)mice with TIMP2 deficiency in their kidneys were generated,and the renal function as well as tissue sections of mice were evaluated,so as to investigate the function of TIMP2 in sepsis-associated AKI.In the in vitro experiments,human kidney(HK-2)cells were stimulated by lipopolysaccharide(LPS)to investigate the mechanism of sepsis-associated AKI.TIMP2 was stably knockdown in HK-2 cells with lentivirus particles expressing specific sh RNA,then flowcytometry and TUNEL assays were performed to assess the influence of TIMP2 and LPS on cell apoptosis.In addition,we detected the expressions of ER stress molecules by q-PCR and western blotting.Stable cell line transfected with TIMP2 overexpression construct was employed to verify its implication in ER stress activation;and we additionally resorted to the specific inhibitor of PERK phosphorylation,called GSK2656157,to establish a rescue experiment.The co-immunoprecipitation assay was performed to find out the relationship between TIMP2 and BiP.We also measured the contents of BiP in the cell culture supernatants and mouse serum by performing enzyme-linked immune-sorbent assay(ELISA).Intracellular BiP and PERK phosphorylation in TIMP2 overexpression cells were evaluated as well,with the additional treatment of protein transport inhibitor,intending to figure out the mechanism of cellular BiP reduction and its impact on ER stress.Results TIMP2 was upregulated in the kidney of mice with sepsis,along with the activation of ER stress.The double-stranded RNA-dependent protein kinase(PKR)-like ER protein kinase(PERK)signaling in ER stress was activated and the expressions of associated apoptotic factors were altered.However,the expression of binding immunoglobulin protein(BiP)was decreased while its m RNA level was not influenced.In addition,TIMP2 knockout significantly alleviated the renal injury induced by sepsis,and suppressed the activation of ER stress.Sepsis-associated renal cell apoptosis was also mitigated in TIMP2 CKO mice.In the in vitro experiments,LPS-induced cell apoptosis was attenuated by TIMP2 knockdown,while overexpression of TIMP2 caused intense activation of PERK signaling in ER stress.TIMP2 mediated apoptosis through the implication of PERK pathway and BCL-2apoptotic signaling.In addition,TIMP2 interacts with BiP;overexpression of TIMP2 inhibited the endoplasmic reticulum retrieval of BiP proteins,resulting in increased secretion of BiP.Conversely,knockdown of TIMP2 suppressed LPS-induced BiP secretion.The reduction of intracellular BiP levels were followed by simultaneous PERK phosphorylation,which initiates the signaling transduction of endoplasmic reticulum stress.Conclusion TIMP2 is involved in the pathological process of septic AKI by mediating endoplasmic reticulum stress and apoptosis.Kidney-specific knockout of TIMP2 alleviates sepsis-associated kidney injury in mice.Intracellular TIMP2 interacts with BiP and then promote its secretion into the extracellular compartment,where the underlying mechanism is related to the endoplasmic reticulum retrieval signaling of BiP.The reduction of intracellular BiP levels results in diminished inhibition of PERK,leading to endoplasmic reticulum stress as well as apoptosis of renal cells.